PF-04691502 is a potent and selective dual PI3K/mTOR (FRAP) inhibitor to phosphorylation of Akt T308 (IC50 = 7.5 nM) and Akt S473 (IC50 = 3.8 nM).[1]
PI3Ks are a family of enzymes involved in cellular functions such as cell growth, proliferation, differentiation, motility, survival and intracellular trafficking, which in turn are involved in cancer.The functions of PI3Ks most relate to the ability of class I PI3K to activate protein kinase B (PKB, aka Akt) as in the PI3K/AKT/mTOR pathway which is an intracellular signaling pathway directly related to cellular quiescence, proliferation, cancer, and longevity. There are many valuable anti-cancer drug treatment targets within this pathway. And also the p110δ and p110γ isoforms regulate different aspects of immune responses. [2]
PF-04691502 is a potential medical drug that functions by inhibiting class I PI3K and mTOR in the PI3K/AKT/mTOR pathway through fluorescence polarization kinase assay, cell,mice and other animal trials, and therefore, through inhibition, results in tumour suppression.[3,4] Short-term exposure to PF-04691502 predominantly inhibits PI3K, whereas mTOR inhibition persists for 24 to 48 hours. PF-04691502 induces cell cycle G(1) arrest, concomitant with upregulation of p27 Kip1 and reduction of Rb. [5] Antitumor activity of PF-04691502 is observed in U87 (PTEN null), SKOV3 (PIK3CA mutation), and gefitinib- and erlotinib-resistant non-small cell lung carcinoma xenografts. PF-04691502 inhibits tumor growth at 7 days by 72%. FDG-PET imaging revealed that PF-04691502 reduces glucose metabolism dramatically. Tissue biomarkers of PI3K/mTOR pathway activity, p-AKT (S473), and p-RPS6 (S240/244), are also dramatically inhibited following PF-04691502 treatment. [6]
Reference:
1.? Yuan J."PF-04691502, a Potent and Selective Oral Inhibitor of PI3K and mTOR Kinases with Antitumor Activity". Mol Cancer Ther, 2011, 10(11), 2189-2199.
2.? Okkenhaug K. "Signaling by the Phosphoinositide 3-kinase Family in Immune Cells.".Annu. Rev. Immunol, 2013. 17 (2): 675–699.
3.? Maira, Sauveur-Michel; Stauffer, Frédéric; Schnell, Christian; García-Echeverría, Carlos. "PI3K inhibitors for cancer treatment: where do we stand ". Biochemical Society Transactions., 2009. 37 (Pt 1): 265–72.
4.? Kinross KM. "In Vivo Activity of Combined PI3K/mTOR and MEK Inhibition in a KrasG12D; Pten Deletion Mouse Model of Ovarian Cancer". Mol Cancer Ther, 2011, 10(8), 1440-1449.
5.? Yuan J, Mol Cancer Ther, 2011, 10(11), 2189-2199
6.? Kinross KM, Mol Cancer Ther, 2011, 10(8), 1440-1449
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Kinase experiment: |
The biochemical protein kinase assays for class I PI3K and mTOR are assessed. The fluorescence polarization assay for ATP competitive inhibition is done as follows: mPI3Kα dilution solution (90 nM) is prepared in fresh assay buffer (50 mM Hepes pH 7.4, 150 mM NaCl, 5 mM DTT, 0.05% CHAPS) and kept on ice. The enzyme reaction contained 0.5 nM mouse PI3Kα (p110α/p85α complex purified from insect cells), 30 μM PIP2, PF-04691502 (0, 1, 4, and 8 nM), 5 mM MgCl2, and 2-fold serial dilutions of ATP (0-800 μM). Final DMSO is 2.5%. The reaction is initiated by the addition of ATP and terminated after 30 minutes with 10 mM EDTA. In a detection plate, 15 uL of detector/probe mixture containing 480 nM GST-Grp1PH domain and 12 nM TAMRA tagged fluorescent PIP3 in assay buffer is mixed with 15 uL of kinase reaction mixture. The plate is shaken for 3 minutes, and incubated for 35 to 40 minutes before reading on an LJL Analyst HT[1]. |
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Cell experiment: |
BT20, U87MG, and SKOV3 cells are plated at 3,000 cell/well in 96-well culture plates in growth medium with 10% FBS. Cells are incubated overnight and treated with DMSO (0.1% final) or serial diluted compound for 3 days. Resazurin is added to 0.1 mg/mL. Plates are incubated at 37°C in 5% CO2 for 3 hours. Fluorescence signals are read as emission at 590 nm after excitation at 530 nm. IC50 values are calculated by plotting fluorescence intensity to drug concentration in nonlinear curves. U87MG and SKOV3 cells are plated in 96-well plates overnight and caspase-3/caspase-7 activity is assessed with the Caspase-Glo 3/7 Assay Kit[1]. |
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Animal experiment: |
Mice[1] Female nu/nu mice (6-8 weeks old) are used. Tumor cells for implantation are harvested and resuspended in serum-free medium mixed with matrigel (1:1). SKOV3, U87MG, or NSCLC cells (2.5-4×106) are implanted subcutaneously into the hind flank region. Treatment started when average tumor size is 100 to 200 mm3. PF-04691502 is formulated in 0.5% methylcellulose in water suspension and given orally once a day. Animal body weights and tumor volumes are measured every 2 to 3 days. Tumor volume is determined with Vernier calipers and calculated. Percentage of tumor growth inhibition (TGI) is calculated. Data are presented as mean±SE. Comparisons between treatment groups and vehicle group are done using 1-way ANOVA by Dunnett's tests. Student's t test is used to determine the P value for the comparison of 2 groups. |
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参考文献: [1]. Yuan J, et al. PF-04691502, a potent and selective oral inhibitor of PI3K and mTOR kinases with antitumor activity. Mol Cancer Ther. 2011 Nov;10(11):2189-99. |
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